1

Optochin-Resistant Streptococcus pneumoniae: Phenotypic and Molecular 1

Characterization of Isolates from Brazil with Description of Five Novel 2

Mutations in the atpC Gene 3

4

Tatiana C.A. Pinto,a Aline R.V. Souza,a Sandrine E.C.M. de Pina,a Natália S. Costa,a 5

Armando A. Borges Neto,a Felipe P.G. Neves,b Vânia L.C. Merquior,c Cícero A.G. 6

Dias,d José M. Peralta,a Lúcia M. Teixeiraa 7

8

Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de 9

Janeiro, RJ 21941-902, Brazila; Instituto Biomédico, Universidade Federal Fluminense, 10

Niterói, RJ 24210-130, Brazilb; Departamento de Microbiologia, Imunologia e 11

Parasitologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ 20551-030, 12

Brazilc; Departamento de Microbiologia e Parasitologia, Universidade Federal de Ciências 13

da Saúde de Porto Alegre, Porto Alegre, RS 90050-170, Brazild 14

15

Address correspondence to Lúcia M Teixeira, lmt2@micro.ufrj.br 16

17

Running Title: Mutations in Optochin-Resistant S. pneumoniae 18

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01168-13 JCM Accepts, published online ahead of print on 24 July 2013

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ABSTRACT 19

Optochin (Opt) susceptibility is largely used for the identification of Streptococcus 20

pneumoniae (Sp) in diagnostic laboratories. Opt-resistant (Optr) Sp isolates have been 21

reported, however, indicating the potential for misidentification of this important pathogen. 22

Point mutations in the atpC gene have been associated with the emergence of Optr Sp but 23

data on the characterization of such atypical variants of Sp are still limited. The present 24

report describes the results of a polyphasic approach to identify and characterize twenty-six 25

Optr Sp isolates recovered from patients or carriers living in Brazil. Sixteen isolates 26

consisted of heterogeneous populations and ten isolates were homogeneously Optr. They 27

had different serotypes and antimicrobial susceptibility profiles. They also presented 28

diverse genetic characteristics, as indicated by PFGE, MLVA and pspA gene typing. Except 29

for Opt MICs (4 to 64-fold higher among Optr variants), Optr and Opts subpopulations 30

originating from the same culture had identical characteristics. Sequencing of the atpC gene 31

of the Optr variants revealed thirteen different nucleotide changes distributed among eight 32

different codons. Changes in codon 49 were the most frequent, suggesting that this might 33

be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel 34

types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, Ala49Gly) 35

were identified. In silico prediction modeling indicated that the atpC gene mutations 36

corresponded to alterations in the transmembrane region of the ATPase, leading to a higher 37

hydrophobicity profile in α-helix 1 and to a lower hydrophobicity profile in α-helix 2. 38

39

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INTRODUCTION 40

Streptococcus pneumoniae (Sp) remains as one of the most important agents of infectious 41

diseases worldwide and a leading cause of invasive and non-invasive infections in children 42

and in the elderly (1). Nasopharyngeal carriage is considered the main reservoir of Sp in 43

nature and a condition of paramount importance for the continuous circulation of this 44

bacterial species among human populations. Due to the significant morbidity and mortality 45

rates associated with pneumococcal diseases, rapid and accurate diagnosis is essential and 46

frequently relies on the isolation and identification of the etiological agent. For this 47

purpose, optochin (Opt) susceptibility testing has been widely used as the primary or even 48

the only test for the presumptive identification of pneumococci, due to the ability to 49

differentiate Sp from the other α-hemolytic streptococci (2-4). 50

Nevertheless, thirty years after the introduction of the Opt susceptibility test as a 51

diagnostic tool (5), the first optochin-resistant (Optr) Sp isolate was described (6). Since 52

then, the isolation of Optr Sp from a variety of clinical sources has sporadically been 53

reported from different locations, with an apparent increasing incidence in the last decade 54

(2, 3, 7-12). The occurrence of such atypical variant is a potential cause of misidentification 55

of this important agent, raising questions about the accuracy of laboratory diagnosis when a 56

single identification procedure based on Opt susceptibility testing is used. 57

Previous studies have shown that single point mutations in the c-subunit (Table 1), 58

and less commonly in the a-subunit, of the atpCAB operon that codes for the molecular 59

target of optochin, the transmembrane F0 F1 ATPase, are present in Optr Sp strains and may 60

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be associated with this unusual phenotype (2, 7, 9, 10, 12-14). Although still uncertain, it 61

has been suggested that use of antimalarial chemotherapy in endemic areas (14), as well as 62

exposition to subinhibitory concentrations of penicillin (15) may be related to the 63

emergence of such mutations. 64

Studies concerning the phenotypic and genetic characteristics of atypical Sp variants 65

expressing resistance to Opt are still limited and may contribute to a better understanding of 66

the mechanisms involved in the emergence, as well as the biological and epidemiological 67

impacts of such resistance trait. The present report describes the results of a polyphasic 68

approach applied to characterize twenty-six Optr Sp isolates recovered from patients and 69

nasopharyngeal carriers living in Brazil. The study encompasses extensive phenotypic and 70

genotypic characterization of Optr Sp isolates presenting a variety of atpC gene mutations, 71

including the description of five novel types of mutations associated with resistance to Opt. 72

Additionally, in silico prediction modeling was performed to evaluate the impact of 73

mutations on the structure and hydrophobicity profile of the c-subunit of ATPase. 74

75

MATERIALS AND METHODS 76

Bacterial strains and identification tests. Twenty-six Optr Sp strains were included in the 77

present study. Among them, four (Sp 910, Sp 913, Sp 917 and Sp 1008) were previously 78

characterized by using a variety of conventional and genetic tests (9). They were included 79

in the present study for evaluation of additional characteristics (determination of Opt MIC, 80

MLVA type and PspA family). All the isolates were recovered during surveillance studies 81

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performed by our group, or were received from different health institutions for 82

confirmation of the identification. Isolates obtained from diseased individuals were 83

recovered from clinical samples taken as part of standard patient care procedures and they 84

do not need an ethical approval for their use. Isolates from carriage studies were recovered 85

from clinical samples collected as approved by the ethics committees of the institutions 86

involved. The isolates were subjected to phenotypic identification tests, including 87

observation of colonial morphology and type of hemolytic activity on blood agar plates; 88

cellular characteristics as observed after Gram stain; optochin susceptibility, bile-solubility, 89

and latex agglutination tests. PCR tests for detection of the lytA, ply and psaA genes were 90

also performed. S. pneumoniae strains ATCC 49619 and ATCC BAA-255 (R6), and S. 91

mitis SS-429 were included as reference strains in all the assays. 92

Optochin susceptibility testing. Opt susceptibility was determined by disk 93

diffusion testing according to standard procedures (4). Optochin disks (BBL Taxo P Discs, 94

BD, Sparks, MD, USA) were applied to the surface of 5% sheep blood agar plates (Plast 95

Labor, Rio de Janeiro, RJ, Brazil) streaked with the isolate being tested. After overnight 96

incubation at 35°C under both 5% CO2 and conventional atmospheres (16), growth 97

inhibition zones around the disks were measured. Isolates displaying inhibition zones ≥14 98

mm in diameter were identified as susceptible, while strains showing zones �14 mm, or 99

zones ≥14 mm but containing colonies inside, were considered resistant. The optochin 100

(Sigma Chemical Co., St. Louis, MO, USA) minimal inhibitory concentration (MIC) values 101

were determined by the agar dilution method (2). Briefly, bacterial suspensions were 102

prepared in 0.9% saline (Sigma) from overnight cultures and adjusted to achieve the 0.5 103

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McFarland turbidity standard. Suspensions were inoculated onto Müeller-Hinton agar 104

containing 5% sheep blood (Plast Labor, Rio de Janeiro, RJ, Brazil) and varying 105

concentrations of optochin (0.5 to 256 μg/mL), followed by incubation at 35°C for 20–24h 106

under 5% CO2 atmosphere. The MIC was defined as the lowest concentration of optochin 107

that inhibited visible growth of the isolate. S. pneumoniae ATCC 49619 and R6 were 108

included as susceptible controls, while S. mitis SS-429 was included as a resistant control in 109

the Opt susceptibility tests. 110

Bile-solubility testing. Bile-solubility tube testing was performed as previously 111

described (4). Briefly, heavy bacterial suspensions were prepared in 0.9% saline (Sigma) 112

from overnight cultures. The suspensions were placed into 2 tubes, and an equal volume of 113

2% deoxycholic acid (Sigma) solution was added to one tube (test tube) while an equal 114

volume of 0.9% saline was added to the other (control tube). Tubes were incubated at 35°C 115

for up to 2 h. Complete visual clearing of the turbidity in the deoxycholate-containing tube, 116

but not in the saline control tube, indicated a positive test. 117

Latex agglutination tests. The isolates were tested for the presence of capsular 118

polysaccharide antigens by latex agglutination testing using the Slidex Pneumo kit 119

according to the manufacturer’s instructions (bioMérieux, Marcy l'Etoile, France). 120

Determination of capsular type. The capsular types were determined either by 121

multiplex PCR using the Latin America scheme (17) or by the standard Quellung reaction 122

with type-specific antisera prepared at the Centers for Disease Control and Prevention 123

(CDC), as earlier described (18). 124

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Antimicrobial susceptibility testing. Antimicrobial susceptibility was evaluated by 125

the agar diffusion method according to the CLSI recommendations and interpretative 126

criteria (19, 20). Antimicrobials tested included chloramphenicol, clindamycin, 127

erythromycin, levofloxacin, oxacillin, rifampicin, trimethoprim/sulfamethoxazole, 128

tetracycline and vancomycin (Oxoid, Basingstoke, Hampshire, United Kingdom). Penicillin 129

MICs were determined by using Etest strips (AB Biodisk, Solna, Stockholm, Sweden). S. 130

pneumoniae ATCC 49619 was used for quality control. 131

Preparation of bacterial DNA. DNAs for all PCR reactions were obtained by 132

using the Chelex® 100 resin (Bio-Rad, Hercules, CA, USA) method. Briefly, a loopful of 133

overnight cultures was suspended in a solution containing 5% Chelex® and proteinase K 20 134

mg/mL (Invitrogen, Life Technologies, Carlsbad, CA, USA). After incubation at 56°C for 1 135

h, followed by incubation at 95°C for 10 min, they were centrifuged and the supernatant 136

was used as the template for all the subsequent PCR reactions. 137

Detection of resistance- and virulence-associated genes. The presence of the 138

macrolide-resistance determinants ermA, ermB and mef (21), the tetracycline-resistance 139

genes tetK, tetL, tetM and tetO (22), as well as the virulence-associated genes ply (coding 140

for pneumolisin), lytA (coding for autolysin) and psaA (coding for pneumococcal surface 141

antigen A) (23, 24) was investigated by PCR using protocols previously described and an 142

automated Veriti 96-well thermal cycler (Applied Biosystems, Inc, Carlsbad, CA, USA). 143

Pulsed-Field Gel Electrophoresis (PFGE) analysis. Genomic DNA was prepared 144

in agarose plugs as described by Teixeira et al. (25), with the following modifications: 145

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bacteria were grown in blood agar plates for 8 to 10 h, plugs were prepared with low-146

melting-temperature agarose at 2.5% (NuSieve GTG agarose, FMC BioProducts, Rockland, 147

ME, USA) and were washed 8 times before restriction. After macrorestriction using SmaI 148

(New England Biolabs, Ipswich, MA, USA), the fragments were separated in a CHEF-DR 149

III system (Bio-Rad) using the parameters recommended by the Pneumococcal Molecular 150

Epidemiology Network (PMEN; http://www.sph.emory.edu/PMEN/index.html). The 151

restriction profiles were analyzed by using the BioNumerics software version 6.6 (Applied 152

Maths, Ghent, East Flanders, Belgium). A dendrogram was created using the unweighted-153

pair group method with arithmetic mean (UPGMA) and the Dice similarity coefficient, 154

with optimization and position tolerance settings of 0.5% and 1.3%, respectively. Profiles 155

showing more than 80% of similarity were considered as belonging to the same cluster. 156

PFGE profiles of the 43 clones described by the PMEN were also included in the in silico 157

analysis for comparative purposes. 158

Multiple Locus VNTR Analysis (MLVA). Eight VNTRs (Spneu15, Spneu17, 159

Spneu25, Spneu33, Spneu36, Spneu37, Spneu39 and Spneu42), selected (N. S. Costa et al., 160

unpublished data) among an initially suggested panel of 18, were amplified by PCR using 161

primers and a protocol described earlier (26). MLVA profiles were analyzed by using 162

BioNumerics software version 6.6 (Applied Maths), in order to construct a dendrogram by 163

the UPGMA and a diagram based on the Minimum Spanning Tree (MST) method. 164

Reference strain S. pneumoniae R6 was used as quality control for the PCR reactions. 165

pspA gene family typing. The pspA genes were detected by PCR using primers and 166

protocols previously described (27). Control strains for pspA family 1 (strains BG9739, 167

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DBL1 and EF10197), family 2 (strains AC122, BG11703, BG7591 and ATCC 6303) and 168

family 3 (strain BG6380) were also included. Strains were categorized as non typeable 169

(NT) if negative results were obtained in 3 attempts of amplification. 170

Sequencing of the atpC gene. The gene coding for the c-subunit of ATPase was 171

amplified by PCR using primers and protocols previously described (2, 9, 13). The 172

fragment was purified using ExoSAP-IT® (USB Affymetrix, Cleveland, OH, USA) and the 173

sequences were obtained using an ABI 3130 Genetic Analyzer (Applied Biosystems). 174

Edition and alignment were performed with the BioEdit software version 7.0.9.0 (28), as 175

well as the construction of charts based on the Kyte & Doolittle Scale Mean 176

Hydrophobicity Profile. Using the amino acid sequences translated from the nucleotide 177

sequences obtained in the study, a predicted model for the c-subunit was designed by using 178

the resources available at the Protein Structure PREDition (PSIPRED) server 179

(http://bioinf.cs.ucl.ac.uk/psipred/; 29). Opts reference strains (S. pneumoniae ATCC 49619 180

and R6) were also included for comparative purposes. 181

182

RESULTS AND DISCUSSION 183

Identification of the isolates and characterization of Opt resistance. The twenty-six 184

Optr Sp isolates included in this study were recovered from different clinically significant 185

sources (14 isolates) or from nasopharyngeal secretions of healthy carriers (12 isolates) 186

between 1995 and 2012, as shown in Fig. 1. All the isolates had the following 187

characteristics: they were gram-positive catalase-negative cocci; presented α-hemolysis on 188

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blood agar plates; gave positive results in the bile solubility and in the latex agglutination 189

tests; possessed the lytA, ply and psaA genes; and were resistant to Opt. 190

Opt susceptibility testing, performed either under CO2-enriched or conventional 191

atmospheres, revealed the occurrence of two phenotypes among the Optr Sp isolates 192

analyzed (Fig. 1). The prevalent phenotype comprised sixteen strains and was expressed as 193

a typical inhibition zone of ≥ 14mm around the Opt disk with colonies inside, representing 194

a heterogeneous population. The subpopulations were discriminated based on the Opt 195

susceptibility and the subsequent experiments were carried out separately for both 196

subpopulations. The second phenotype was observed for the remaining ten Optr Sp strains 197

that had no zone of inhibition around the Opt disk, representing homogeneous resistant 198

populations. The finding of two different Optr phenotypes among Sp isolates has been 199

reported before, among isolates from Portugal (11) and from the United States (2) with 200

different distributions. Among isolates from Portugal, a predominance of homogeneous 201

Optr populations was observed, while the majority of the isolates from the United States 202

were reported to be composed of heterogeneous populations as also observed in the present 203

study. Opt MICs ranged from 8 to 64 μg/mL for the Optr Sp strains (Fig. 1), which is in 204

agreement with earlier reports on Opt resistance (2, 7, 11). On the other hand, the sixteen 205

Opts subpopulations derived from heterogeneous cultures, as well as the susceptible 206

reference strains S. pneumoniae ATCC 49619 and R6, showed MIC values of 1 to 2 207

μg/mL, which were 4- to 64-fold lower than those obtained for their respective Optr 208

counterparts. Except for the unusual Optr phenotype, all the Sp strains analyzed showed 209

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phenotypic and genetic characteristics of typical pneumococcal isolates, as they were bile-210

soluble, produced capsular antigens, and carried the ply, lytA and psaA genes. 211

Distribution of capsular types. The isolates tested belonged to a variety of 212

capsular types (Fig. 1). Most of the sixteen different capsular types (1, 6A, 6B, 6C, 9N, 213

10A, 11A, 12F, 13, 14, 15C, 16F, 18C, 19A, 23A, 23F) observed among the isolates 214

included in the present study correspond to types commonly found in our region (30, 31), 215

reinforcing the concept that serotype distribution among Optr Sp strains is mostly dependent 216

on the epidemiological profile of each area (2, 3, 10-12). Identical serotypes were observed 217

for the Optr and Opts subpopulations derived from the same culture. Interestingly, among 218

the eight isolates belonging to serogroup 6, three belonged to the recently described variant 219

6C (32), that seems to have emerged during the post-vaccination era in developed countries 220

(33, 34). Moreover, five of the capsular types identified (1, 6B, 14, 18C and 23F), 221

comprising a total of ten strains, are included in the 10-valent pneumococcal conjugate 222

vaccine (PCV-10), that has been incorporated into the Brazilian Immunization Programme 223

in 2010 (http://portal.saude.gov.br/portal/saude/visualizar_texto.cfm?idtxt=21462). 224

Antimicrobial susceptibility profiles. The Optr Sp strains were susceptible to most 225

of the antimicrobial agents tested (Fig. 1) with only two of them presenting a multidrug-226

resistance profile (including simultaneous resistance to clindamycin, erythromycin, 227

penicillin, tetracycline and trimethoprim/sulfamethoxazole), suggesting that Opt resistance 228

is not associated with increasing or specific resistance to other drugs. Resistance to both 229

erythromycin and clindamycin was detected in 2 (7.7%) strains, and it was associated with 230

the simultaneous presence of the ermB and mef genes. Resistance to tetracycline was 231

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observed in four (15.4%) isolates carrying the tetM gene. All the resistance-associated 232

genes detected are commonly found among conventional Opts Sp strains as well (35). A 233

total of five (19.2%) isolates showed non-susceptibility to penicillin, displaying MICs 234

ranging from 0.32 to 2 μg/mL. Isolates belonging to serotypes 14 and 6B, recovered from 235

blood or sputum, had the higher penicillin MICs (0.5 or 2 μg/mL), while serotypes 18C and 236

19A isolates, obtained from nasopharynx or cerebrospinal fluid (CSF), had the lower MICs 237

(0.32 or 0.38 μg/mL). As observed for serotype distribution, antimicrobial susceptibility 238

among Optr Sp isolates included in this study is probably reflecting the epidemiological 239

profile of the region. In addition, Optr and Opts subpopulations originating from the same 240

culture showed identical profiles of antimicrobial susceptibility. 241

Genetic diversity evaluated by PFGE, MLVA and pspA gene family typing. 242

PFGE (Fig. 1) and MLVA (Fig. 2) revealed a high degree of genetic diversity, generating 243

26 and 25 different profiles, respectively, among the twenty-six Optr isolates analyzed, and 244

indicating that Opt resistance is not due to clonal dissemination, in accordance with data 245

from other regions (2, 3, 9, 10, 12). Moreover, PFGE revealed that five Optr isolates were 246

respectively related to five internationally disseminated clones recognized by the PMEN 247

(http://www.sph.emory.edu/PMEN/index.html) (Fig. 1). Among them, four (Spain6B-2, 248

Spain9V-3, Colombia23F-26 and Sweden1-40) are known to be prevalent in Brazil (31, 36, 249

37). Except for one, those five Optr Sp strains were recovered from cases of invasive 250

pneumococcal disease, and two of them were non-susceptible to penicillin, with one being 251

multidrug resistant. Likewise, the occurrence of strains related to some of the PMEN 252

clones, including Spain9V-3 and Colombia23F-26, were also reported among Portuguese Optr 253

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Sp isolates (3, 11). Heterogeneity was also revealed by analysis of the pspA genes. Thirteen 254

Optr Sp isolates were typed as pspA family 1, while ten belonged to pspA family 2. The 255

pspA family 3 was observed for two strains and one strain had no detectable pspA genes 256

(Fig. 1). These results show that distribution of pspA gene families among Optr Sp strains 257

follows a profile quite similar to that seen among Sp isolates in general, with predominance 258

of pspA families 1 and 2 (38). Moreover, Opts subpopulations derived from heterogeneous 259

cultures showed PFGE profiles, MLVA types and pspA gene family types identical to their 260

respective Optr counterparts. Although this is, to our knowledge, the first report on the use 261

of MLVA and pspA gene typing to characterize Optr isolates, indistinguishable PFGE 262

profiles between Optr and Opts subpopulations deriving from a single culture have been 263

observed by others (2, 8, 11). 264

Nucleotide sequences of the atpC gene. Sequencing of the atpC gene, which codes 265

for the c-subunit of the pneumococcal ATPase, revealed single-base substitutions leading to 266

amino acid modifications in all the twenty-six Optr Sp strains analyzed (Fig. 1). Thirteen 267

different nucleotide changes distributed among eight different codons were observed (see 268

Fig. S1 in the supplemental material). Five of them, comprising six strains, consisted of 269

novel alterations: Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly. Among them, 270

three mutation locations (codons 13, 18 and 31) were never reported before among Optr Sp 271

isolates obtained from clinical sources. A summary with all the mutations described among 272

Optr Sp strains until this date is presented in Table 1, including those described for the first 273

time in the present study. Mutations obtained by in vitro transformation or induction 274

experiments (10, 14) were not included. 275

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The mutations observed were randomly distributed among Optr Sp strains derived 276

from either heterogeneous or homogeneous populations (Fig. 1). Therefore, no correlation 277

was observed between the Optr phenotype and the type of mutation. Likewise, no 278

correlation with Opt MICs was noted either. On the other hand, no mutations were 279

observed in the atpC sequences of Opts subpopulations derived from heterogeneous 280

cultures, as their sequences were indistinguishable from those obtained for S. pneumoniae 281

strains ATCC 49619 and R6 (see Figure S1 in the supplemental material), reinforcing the 282

association between atpC mutations and Opt resistance. The fact that Optr and Opts 283

subpopulations were only differentiated on the basis of the Opt MICs and the atpC gene 284

sequencing suggests that an originally homogeneous Opts population might have undergone 285

a point mutation resulting in two different types of cells. 286

The c-subunit of the ATPase is known to consist of 66 amino acids distributed in 287

two antiparallel α-helixes linked by a conserved cytoplasmic loop (13). Previous studies 288

have shown that mutations leading to Opt resistance usually occur in the transmembrane 289

portion of the α-helixes, as it does not interfere with the activity of the whole enzyme (14). 290

Indeed, all the altered codons identified in this study are located in the transmembrane 291

region according to the predicted model of the subunit (Fig. 3). Eleven strains presented 292

alterations in the α-helix 1 of the c-subunit (codons 13, 14, 18, 20, 23 or 31) and fifteen in 293

the α-helix 2 (codons 45 or 49), among which twelve isolates (accounting for 48% of all 294

Optr Sp strains analyzed) had mutations in codon 49 (Table 1 and Fig. 1). 295

It was noticeable that all the atpC gene mutations identified in the present study 296

generated codifying sequences, involving amino acid replacement, and no termination 297

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codons that would inhibit expression of atpC due to early termination were detected. Single 298

base substitutions are likely to occur randomly along the atpC gene sequence over time, 299

including synonymous or even deleterious mutations. However, those that are evolutionary 300

neutral, or maybe advantageous, and that lead to phenotypic variation, as illustrated by 301

optochin resistance, are possibly the only ones eventually detected. 302

The prevalence of mutations in codon 49 (Table 1) is noteworthy, leading to 303

speculations on its role as a hot-spot for mutations associated with resistance to Opt. No 304

peculiar characteristics around codon 49 that could predispose this part of the atpC gene to 305

frequent mutations, such as a higher incidence of repeated nucleotides, were observed in 306

the present work. On the other hand, in the predicted folded structure of ATPase, certain 307

amino acids, including those designated by codon 49, are placed next to residues of 308

glutamic or aspartic acid, two non-essential amino acids, that are believed to represent the 309

central portion of α-helix 2 and the binding site for optochin molecules (13). In terms of 310

natural selection and evolution, especially when considering optochin and its analogues as 311

selective pressures, codons producing such amino acids, as codon 49, could represent 312

preferential positions for preserved non-synonymous genetic alterations associated with 313

phenotypic advantages. 314

Although the exact mechanisms leading to Opt resistance remain to be elucidated, it 315

is reasonable to infer that structural alterations in the folding of the protein generated as a 316

result of a mutated atpC sequence, rather than the alterations in the amino acid sequences 317

solely, may, at least in part, account for resistance to optochin. It has also been suggested 318

that the alterations probably change the hydrophobicity profile of the molecule, causing the 319

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loss of affinity to the drug (14). Indeed, among Optr Sp strains included in the present 320

study, changes in the α-helix 1 led, in general, to substitutions for amino acids with higher 321

hydrophobicity profiles (Fig. 4A), while in the α-helix 2 the replacements were for amino 322

acids with lower hydrophobicity profiles (Fig. 4B). Additional studies, however, are 323

required to elucidate the direct or indirect consequences of these changes in the interaction 324

with optochin. 325

Our data indicate that emergence of Optr Sp strains is not due to a clonal spread, 326

since they may possess a diversity of serotypes, phenotypes and genotypes. Also, it seems 327

to be related to a random mutational event, which is more likely to occur in the c-subunit of 328

ATPase, and more frequently in codon 49, near the center of the α-helix 2. The occurrence 329

of two types of Optr Sp strains additionally increases the complexity of this phenomenon, 330

although their individual importance or consequences are still unclear. Overall, our results 331

provide additional information and reinforce previous observations about Opt resistance 332

among Sp strains, contributing to the global and local pool of data on the characteristics of 333

these atypical isolates. Healthcare-associated professionals should be aware of the 334

occurrence of the unusual Optr Sp strains, especially considering that they can be isolated 335

from a variety of clinical sources, including asymptomatic carriers and patients with 336

invasive and non-invasive infections, leading to incorrect bacterial identification, and 337

ultimately to misinterpretation of carriage studies or imprecise diagnosis and treatment of 338

infectious conditions. 339

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Nucleotide sequence accession numbers. The atpC gene sequences of the 22 340

isolates originally reported in the present study were deposited in the GenBank database 341

under accession numbers KC513927 to KC513948, as indicated in Fig. 2A. 342

343

ACKNOWLEDGMENTS 344

This work was supported in part by Coordenação de Aperfeiçoamento de Pessoal de Nível 345

Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico 346

(CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and 347

Ministério da Ciência e Tecnologia (MCT/PRONEX), Brazil. The authors would like to 348

thank Dr. Maria Cristina C. Brandileone (Instituto Adolfo Lutz, SP, Brazil) for providing 349

most of the control strains used in the pspA gene typing experiments, and Dr. Lesley 350

McGee (Centers for Disease Control and Prevention, Atlanta, GA, USA) for providing 351

representative strains of the PMEN clones. The authors declare that they have no competing 352

interests. 353

354

REFERENCES 355

1. Tan TQ. 2012. Pediatric invasive pneumococcal disease in the United States in the era of 356

conjugate pneumococcal vaccines. Clin. Microbiol. Rev. 25:409-419. 357

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TABLE 1 Mutations in the atpC gene described to date among optochin-resistant Streptococcus pneumoniae isolates 480

Target codon (amino acid alteration)a Number of strains reported Country of origin (reference)

13 (Met→Ile) 1 Brazil (This study)

14 (Gly→Ser) 2 Brazil (This study); France (7)

18 (Gly→Ser) 2 Brazil (This study)

20 (Gly→Ser) 3 Brazil (This study); United States (2)

20 (Gly→Ala) 1 Brazil (This study)

23 (Met→Ile) 4 Brazil (9, this study); United States (2)

31 (Ala→Val) 1 Brazil (This study)

44 (Met→Ile) 1 Japan (12)

45 (Phe→Leu) 2 Brazil (9, this study)

45 (Phe→Val) 1 Brazil (This study)

47 (Gly→Val) 1 Argentina (10)

48 (Val→Phe) 3 Japan (12)

49 (Ala→Ser) 10 Argentina (10); Brazil (9, this study); Japan (12)

49 (Ala→Thr) 10 Argentina (10); Brazil (This study); Japan (12); United States (2)

49 (Ala→Gly) 1 Brazil (This study)

50 (Phe→Leu) 1 Japan (12)

aSubstitutions first and only detected in Brazil are outlined in bold. 481

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LEGENDS TO FIGURES 483

484

FIG 1 Genetic relationship among optochin-resistant Streptococcus pneumoniae (Optr Sp) isolates 485

included in the study as evaluated by PFGE analysis. The 80% cut-off considered for clonal groups 486

delineation is illustrated by a red dotted line. 487 aStrain identification; those highlighted in red are reference strains of internationally disseminated 488

clones that were genetically related to some of the Optr Sp strains. 489 bSource of isolation: NP, nasopharynx; U, unknown; LRT, lower respiratory tract; CSF, 490

cerebrospinal fluid; ED, eye discharge. 491 cOptochin susceptibility phenotype: HE, heterogeneous population; HO, homogeneous population 492 dpspA gene family: NT, non typeable. 493 eMinimal inhibitory concentration (MIC) of optochin expressed in μg/mL. 494 fMutation in the atpC gene, including codon number and the respective deduced amino acid 495

substitution. 496 gSXT, trimethoprim/sulfamethoxazole; PEN, penicillin; ERY, erythromycin; CLI, clindamycin; 497

TET, tetracycline; CHL, chloramphenicol. 498

499

500

501

FIG 2 Genetic relationship among optochin-resistant Streptococcus pneumoniae (Optr Sp) 502

isolates included in this study as evaluated by MLVA analysis. A. Dendrogram showing the 503

genetic relatedness, the alleles detected for each VNTR, and the GenBank accession 504

numbers (a) of the atpC gene sequences of the 26 Optr Sp strains. The 80% cut-off 505

considered for clonal groups delineation is illustrated by a red dotted line. B. Diagram 506

constructed by using the Minimum Spanning Tree (MST) method. Node colors represent 507

the different serotypes listed in Fig 1. Higher levels of genetic relationship between nodes 508

are indicated by darker lines. 509

510

511

512

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FIG 3 Predicted model of the c-subunit of ATPase based on the atpC gene sequences 513

obtained in this study. A. Protein secondary structure prediction scheme obtained by using 514

the Protein Secondary PREDiction (PSIPRED) method. Amino acids showing alterations 515

in the present study are highlighted with red boxes. B. Transmembrane protein structure 516

prediction cartoon obtained by using the MEMbrane protein Structure And Topology 517

(MEMSAT) method. S1, helix 1; S2, helix 2. The illustrations shown were designed using 518

amino acid sequence of strain Sp 1835, since all the optochin-resistant Streptococcus 519

pneumoniae isolates included in this study generated identical predictions. 520

521

522

523

FIG 4 Hydrophobicity profiles of the c-subunit of ATPase of optochin-resistant 524

Streptococcus pneumoniae (Optr Sp) isolates. A. Mean hydrophobicity profiles obtained for 525

seven representative Optr Sp isolates with alterations in six different codons belonging to 526

the helix 1 of c-subunit of ATPase. B. Mean hydrophobicity profiles obtained for five 527

representative Optr Sp isolates with alterations in two different codons belonging to the 528

helix 2 of c-subunit of ATPase. Reference strain ATCC 49619 was also included. Codons 529

with alterations are outlined with a vertical black line. 530

531

532

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